We have adapted the methods for absolute quantification based on the targeted proteomics combined with the use of peptide standards and used the data for robust predictive modeling of the signaling pathways in the immune system. We used osteoclast development from macrophages as the initial experimental model. We used the well characterized murine monocyte-macrophage RAW 264.7 cell line as the osteoclast precursor model cell line. The cells fuse to form multinucleated osteoclasts when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL), but the differentiation process is inhibited by sphingosine-1 -phosphate (S1P). The mRNA levels of many proteins change and we wanted to see if these changes are reflected in changes of the cell proteome. We have optimized cell culture conditions and methods for osteoclast enrichment. Using SILAC (stable isotope labeling with amino acids in cell culture) we compared the proteomes of untreated RAW 264.7 cells, intermediate osteoclasts and differentiated, multinucleated osteoclasts. The analysis revealed a set of differentially expressed proteins, which we used to design a set of standard peptides for absolute quantification by mass spectrometry. We have also performed mRNA expression analysis using microarrays and identified major differences between all three cell types. We found that compared to osteoclast precursors, multinucleated osteoclasts conserve energy by down-regulating pathways involved in cell cycle control, gene expression, and protein synthesis. In agreement with previous reports, multinucleated osteoclasts were found to express relatively high levels of V-ATPase, TRAP, cathepsin K, and integrins. Proteins involved in ATP synthesis and catabolism, localized primarily in the mitochondria, were also upregulated in multinucleated osteoclasts, suggesting that osteoclasts up-regulate ATP production compared with osteoclast precursors and intermediate osteoclasts. We have confirmed that both mitochondrial mass and potential are elevated in mature osteoclasts, and median mitochondrial protein expression was significantly higher than the median protein expression in other organelles (1). S1P regulates the chemoattraction and chemorepulsion of osteoclast precursors to and from bones. The murine macrophage RAW 264.7 cells, used here as a model, express two receptors for S1P: S1PR1 and S1PR2. These receptors have markedly different affinity to S1P and cause the opposite effects upon exposure to low/high concentrations of S1P. To develop a deeper understanding of mammalian cell chemotaxis, we used transcriptomics, shotgun proteomics, targeted proteomics, and pathway simulation to investigate S1P-mediated chemotaxis of osteoclast precursors. Transcriptomics using RNA-seq enabled the identification and quantitation of RNA transcripts and shotgun proteomics enabled the identification of proteotypic peptides selected based on peptide proteotypic qualities, sequence uniqueness, and vulnerability to modification (e.g., oxidation and deamidation), eliminating many theoretically possible peptides, which could be non-compatible with mass spectrometric analysis. We used the quantitative data obtained from osteoclast precursors by shotgun proteomics to find the peptides amenable to analysis in our Orbitrap Velos. SPOT synthesis was used to prepare a set of 409 standard, synthetic peptides, which we used to assess the protein expressions in macrophages. Single Reaction Monitoring (SRM) of RAW264.7 cell lysates spiked with the standard peptides resulted in the confident identification and semi-quantitation of 208 of the 409 peptide targets from proteins in the chemokine signaling network. The SRM analysis of a smaller set of 65 heavy-labeled, quantitated internal peptide standards from proteins differentially expressed under different experimental conditions provided absolute numbers of molecules. These data were then used to design targeted proteomics assays of the proteins of the mouse chemotaxis pathway. Targeted proteomics assays using nano-flow liquid chromatography coupled to selected reaction monitoring mass spectrometry (LC-SRM) were performed to produce absolute abundance values (in units of copies/cell) for each of the target proteins within RAW 264.7 cells. RAW cells were again used as model osteoclast precursors because they have very similar S1P-directed chemotaxis behavior. Rules-based pathway modeling enabled the simulation of the mouse chemotaxis pathway based on bi-molecular interactions within the geometry of a three-dimensional in silico RAW cell. Measured protein abundance values, used as simulation input parameters, led to in silico pathway behavior matching in vitro measurements. Moreover, once model parameters were established, even simulated responses towards stimuli that were not used for parameterization were consistent with experimental findings. These findings demonstrated the feasibility and value of combining targeted mass spectrometry with pathway modeling for advancing biological insight and defined our experimental approach to modeling other immune system signaling pathways (2, 3). In the TLR signaling network modeling study, we utilize targeted proteomics with transcriptomics to aid in contructing a computational model of the LPS-TLR4 signaling pathway in a mouse monocyte-macrophage cell line RAW264.7. A set of protein targets was identified from a review of current literature and KEGG pathways describing LPS-TLR4 signaling. Corresponding peptides were selected after scoring based on several criteria including length, shotgun proteomics identification, and potential PTM sites as determined by literature mining by motif prediction (Pubmed). Peptides were analyzed in both shotgun-mode and SRM-mode to determine the potential for success in biological samples. RAW cell samples stimulated with LPS for different times were analyzed for the selected peptides. We performed semi-quantitative analysis with the external peptide standards and obtained proteotypic peptides for most of the proteins in the canonical TLR signaling network. Based on these results, we have obtained and heavy-labeled internal peptide standards against corresponding protein targets for absolute quantitation measurements. Additionally, we designed, obtained and tested a set of peptides phosphorylated at the crucial regulatory residues of the proteins in the TLR signaling network. We have performed robust quantitative measurements with the heavy-labeled peptide standards spiked into the cell lysates. We examined unstimulated controls and cells stimulated with LPS for 30 minutes and obtained absolute protein measurements for both. In collaboration with Drs. Martin Meier-Schellersheim and Bastian Angermann, we have created the network of essential proteins and their interactions for the Simmune-based model and began modeling the network changes following TLR stimulation with LPS. The model will incorporate also the measurements of PTM changes obtained from project AI001084-07 and the binding constants we are getting using the modeling with protein structure data. In this project, we are able to reach beyond basal level quantification to further develop and test the TLR signaling network model under a variety of biologically relevant perturbations (different and modified TLR ligands, whole pathogens, and cells with mutations in specific signaling molecules). 1. An E, Narayanan M, Manes NP, and Nita-Lazar A. (2014) Mol Cell Proteomics 2014 Oct;13(10):2687-704. doi: 10.1074/mcp.M113.034371 2. Manes NP, Angermann BR, Koppenol-Raab M, An E, Sjoelund VH, Sun J, Ishii M, Germain RN, Meier-Schellersheim M, and Nita-Lazar A. (2015) Mol Cell Proteomics. 2015 Oct;14(10):2661-81. doi: 10.1074/mcp.M115.048918. 3. Manes NP, Mann JM, and Nita-Lazar A. (2015) J Vis Exp 102, doi: 10.3791/529